This invention relates to a series of substituted xcex1-aminosulphonyl-acetohydroxamic acids which are inhibitors of zinc-dependent metalloprotease enzymes. In particular, the compounds are inhibitors of certain members of the matrix metalloprotease (MMP) family.
Matrix metalloproteases (MMPs) constitute a family of structurally similar zinc-containing metalloproteases, which are involved in the remodelling and degradation of extracellular matrix proteins, both as part of normal physiological processes and in pathological conditions. Since they have high destructive potential, MMPs are usually under close regulation and failure to maintain MMP regulation may be a component of a number of diseases and pathological conditions, including atherosclerotic plaque rupture, heart failure, restenosis, periodontal disease, tissue ulceration, wound repair, cancer metastasis, tumour angiogenesis, age-related macular degeneration, fibrotic disease, rheumatoid arthritis, osteoarthritis and inflammatory diseases dependent on migratory inflammatory cells.
Another important function of certain MMPs is to activate various enzymes, including other MMPs, by cleaving the pro-domains from their protease domains. Thus some MMPs act to regulate the activities of other MMPs, so that over-production of one MMP may lead to excessive proteolysis of extracellular matrix by another. Moreover, MMPs have different substrate preferences (shown in the following Table for selected family members) and different functions within normal and pathological conditions. For recent reviews of MMPs, see Current Pharmaceutical Design, 1996, 2, 624 and Exp. Opin. Ther. Patents, 1996, 6, 1305.
Excessive production of MMP-3 is thought to be responsible for pathological tissue breakdown which underlies a number of diseases and conditions. For example, MMP-3 has been found in the synovium and cartilage of osteoarthritis and rheumatoid arthritis patients, thus implicating MMP-3 in the joint damage caused by these diseases: see Biochemistry, 1989, 28, 8691 and Biochem. J., 1989, 258, 115. MMP-13 is also thought to play an important role in the pathology of osteoarthritis and rheumatoid arthritis: see Lab. Invest., 1997, 76, 717 and Arthritis Rheum., 1997, 40, 1391. The compounds of the present invention inhibit both MMP-3 and MMP-13 and thus may be of utility in treating these diseases.
The over-expression of MMP-3 is also thought to be responsible for much of the tissue damage and chronicity of chronic wounds, such as venous ulcers, diabetic ulcers and pressure sores: see Brit. J. Dermatology, 1996, 135, 52.
Furthermore, the production of MMP-3 may also cause tissue damage in conditions where there is ulceration of the colon (as in ulcerative colitis and Crohn""s disease: see J. Immunol., 1997 158, 1582 and J. Clin. Pathol., 1994, 47, 113) or of the duodenum (see Am. J. Pathol., 1996, 148, 519).
Moreover, MMP-3 may also be involved in skin diseases such as dystrophic epidermolysis bullosa (see Arch. Dermatol. Res., 1995, 287, 428) and dermatitis herpetiformis (see J. Invest. Dermatology, 1995, 105, 184).
Finally, rupture of atherosclerotic plaques by MMP-3 may lead to cardiac or cerebral infarction: see Circulation, 1997, 96, 396. Thus, MMP-3 inhibitors may find utility in the prevention of heart attack and stroke.
Studies of human cancers have shown that MMP-2 is activated on the invasive tumour cell surface (see J. Biol.Chem., 1993, 268, 14033) and BB-94, a non-selective peptidic hydroxamate MMP inhibitor, has been reported to decrease the tumour burden and prolong the survival of mice carrying human ovarian carcinoma xenografts (see Cancer Res., 1993, 53, 2087). Certain compounds of the present invention inhibit MMP-2 and therefore may be useful in the treatment of cancer metastasis and tumour angiogenesis.
Various series of MMP inhibitors have appeared in the patent literature. For example, xcex1-arylsulphonamido-substituted acetohydroxamic acids are disclosed in EP-A-0606046, WO-A-9627583 and WO-A-9719068, whilst EP-A-0780386 discloses certain related sulphone-substituted hydroxamic acids.
The compounds of the present invention are inhibitors of some of the members of the MMP family. In particular, they are potent inhibitors of MMP-3 and MMP-13, with certain compounds exhibiting varying degrees of selectivity over other MMPs, such as MMP-1, MMP-2 and MMP-9. Certain of the compounds are potent MMP-2 inhibitors.
Thus, according to the present invention, there is provided a compound of formula (I): 
or a pharmaceutically or veterinarily acceptable salt thereof, or a pharmaceutically or veterinarily acceptable solvate (including hydrate) of either entity,
wherein
the broken line represents an optional bond;
A is C or CH;
B is CH2, O or absent;
R1 and R2 are each independently selected from hydrogen, C1 to C6 alkyl optionally substituted with C1 to C4 alkoxy or phenyl, and C1 to C6 alkenyl; or, together with the carbon atom to which they are attached, form a C3 to C6 cycloalkyl group which optionally incorporates a heteroatom linkage selected from O, SO, SO2 and
NR6 or which is optionally benzo-fused;
R3 is hydrogen, halo, R7 or OR7;
R4 is hydrogen, C1 to C4 alkyl, C1 to C4 alkoxy, trifluoromethyl or halo;
R6 is hydrogen or C1 to C4 alkyl;
R7 is a monocyclic or bicyclic ring system selected from phenyl, thienyl, furyl, pyridinyl, pyrimidinyl, naphthyl, indanyl, benzothienyl, benzofuranyl, 2,3-dihydrobenzofuranyl, indolyl, quinolinyl, isoquinolinyl, benzodioxolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl and benzodioxanyl, any of which ring systems is optionally substituted with one or two substituents selected from C1 to C4 alkyl optionally substituted with C1 to C4 alkoxy or hydroxy, C1-C4 alkoxy optionally substituted with C1 to C4 alkoxy or hydroxy, C1 to C4 alkylthio, trifluoromethyl, trifluoromethoxy, halo and cyano;
m is 1or 2; and
n is 0, 1 or 2;
with the proviso that B is not O when A is C.
In the above definition, unless otherwise indicated, alkyl, alkoxy, alkylthio and alkenyl groups having three or more carbon atoms may be straight chain or branched chain. Halo means fluoro, chloro, bromo or iodo.
The compounds of formula (I) may contain one or more chiral centres and therefore can exist as stereoisomers, i.e. as enantiomers or diastereoisomers, as well as mixtures thereof. The invention includes both the individual stereoisomers of the compounds of formula (I) and any mixture thereof. Separation of diastereoisomers may be achieved by conventional techniques, e.g. by fractional crystallisation or chromatography (including HPLC) of a diastereoisomeric mixture of a compound of formula (I) or a suitable salt or derivative thereof. An individual enantiomer of a compound of formula (I) may be prepared from a corresponding optically pure intermediate or by resolution, either by HPLC of the racemate using a suitable chiral support or, where appropriate, by fractional crystallisation of the diastereoisomeric salts formed by reaction of the racemate with a suitable optically active base or acid.
Furthermore, compound of formula (I) which contain alkenyl groups can exist as cis-stereoisomers or trans-stereoisomers. Again, the invention includes both the separated individual stereoisomers as well as mixtures thereof.
Also included in the invention are radiolabelled derivatives of compounds of formula (I) which are suitable for biological studies.
Compounds of formulae (I) may provide pharmaceutically or veterinarily acceptable base salts, in particular non-toxic alkali metal salts, with bases. EXAMPLEs include the sodium and potassium salts. The pharmaceutically or veterinarily acceptable salts of the compounds of formula (I) which contain a basic centre are, for example, non toxic acid addition salts formed with inorganic acids such as hydrochloric, hydrobromic, sulphuric and phosphoric acid, with organo-carboxylic acids, or with organo-sulphonic acids.
A preferred group of compounds of formula (I) is that wherein B is absent; R1 is hydrogen, C1 to C4 alkyl optionally substituted with methoxy or phenyl, or C1 to C5 alkenyl; R2 is hydrogen or C1 to C4 alkyl; or R1 and R2, together with the carbon atom to which they are attached, form a C4 to C5 cycloalkyl group which optionally incorporates a heteroatom linkage selected from O and NR6 or which is optionally benzo-fused; R3 is selected from 4-phenyl, 4-pyridinyl, 4-(indan-5-yl), 4-(2,3-dihydrobenzofuran-5-yl), 4-(quinolin-3-yl), 4-(benzodioxol-5-yl) and 4-(benzimidazol-5-yl), any of which is optionally substituted with one or two substituents selected from C1 to C3 alkyl optionally substituted with methoxy or hydroxy, C1 to C3 alkoxy optionally substituted with methoxy or hydroxy, methylthio, trifluoromethyl, trifluoromethoxy, fluoro, chloro and cyano; R4 is hydrogen, methyl, ethyl, methoxy, trifluoromethyl, fluoro or chloro; R6 is methyl; m is 2; and n is 1.
A more preferred group of compounds of formula (I) is that wherein R1 is hydrogen, methyl, ethyl, 2-methylprop-1-yl, but-1-yl, 2-methoxyethyl, benzyl, 3-phenylprop-1-yl, allyl, 2-methylallyl, 3,3-dimethylallyl; R2 is hydrogen, methyl or ethyl; or R1 and R2, together with the carbon atom to which they are attached, form a cyclobutyl, cyclopentyl, tetrahydropyran-4,4-diyl, 1-methylpiperidin-4,4-diyl or indan-2,2-diyl group; R3 is 4-phenyl, 4-(2-methylphenyl), 4-(3-methylphenyl), 4-(3-ethylphenyl), 4-[3-(prop-2-yl)phenyl], 4-(3,5-dimethylphenyl), 4-(3-methoxymethylphenyl), 4-(3-hydroxymethylphenyl), 4-(2-methoxyphenyl), 4-(3-methoxyphenyl), 4-(3-ethoxyphenyl), 4-(4-ethoxyphenyl), 4-[3-(prop-1-oxy)phenyl], 4-[3-(prop-2-oxy)phenyl], 4-[4-(prop-2-oxy)phenyl], 4-(3,4-dimethoxyphenyl), 4-[3-(2-methoxyethoxy)phenyl], 4-[3-(2-hydroxyethoxy)phenyl], 4-(3-methylthiophenyl), 4-(3-trifluoromethylphenyl), 4-(3-trifluoromethoxyphenyl), 4-(2-fluorophenyl), 4-(3-chloro-4-fluorophenyl), 4-(3-cyanophenyl), 4-(pyridin-2-yl), 4-(pyridin-3-yl), 4-(pyridin4-yl), 4-(6-ethoxypyridin-2-yl), 4-(5-ethoxypyridin-3-yl), 4-(indan-5-yl), 4-(2,3-dihydrobenzofuran-5-yl), 4-(quinolin-3-yl), 4-(benzodioxol-5-yl), 4-(2,2-dimethylbenzodioxol-5-yl) and 4-(1,2-dimethylbenzimidazol-5-yl); and R4 is hydrogen, 2-methyl, 3-methyl, 3-ethyl, 3-methoxy, 3-trifluoromethyl, 3-fluoro or 3-chloro.
A particularly preferred group of compounds of formula (I) is that wherein R1 and R2 are both hydrogen or methyl or, together with the carbon atom to which they are attached, form a cyclobutyl, cyclopentyl, tetrahydropyran-4,4-diyl or 1-methylpiperidin-4,4-diyl group; R3 is 4-phenyl, 4-(3-methoxyphenyl), 4-(3-ethoxyphenyl), 4-[3-(2-methoxyethoxy)phenyl], 4-[3-(2-hydroxyethoxy)phenyl] or 4-(6-ethoxypyridin-2-yl); and R4 is 3-methyl or 3-methoxy.
Especially preferred individual compounds of the invention include
N-hydroxy-2-{4-[4-(3-ethoxyphenyl)-3-methylphenyl]-1,2,3,6-tetrahydropyridin-1-ylsulphonyl}acetamide;
N-hydroxy-2-{4-[4-(3-ethoxyphenyl)-3-methylphenyl]-1,2,3,6-tetrahydropyridin-1-ylsulphonyl}-2-methylpropanamide;
N-hydroxy-2-{4-[4-(3-ethoxyphenyl)-3-methylphenyl]piperidin-1-ylsulphonyl}-2-methylpropanamide;
N-hydroxy-1-{4-[4-(3-methoxyphenyl)-3-methylphenyl]piperidin-1-ylsulphonyl}cyclopentanecarboxamide;
N-hydroxy-1-{4-[4-(3-methoxyphenyl)-3-methylphenyl]piperidin-1-ylsulphonyl}cyclobutanecarboxamide;
N-hydroxy-2-{4-[4-(3-ethoxyphenyl)-3-methoxyphenyl]piperidin-1-ylsulphonyl}-2-methylpropanamide;
N-hydroxy-2-{4-[4-(6-ethoxypyridin-2-yl)-3-methylphenyl]piperidin-1-ylsulphonyl}-2-methylpropanamide;
N-hydroxy-2-{4-[4-(3-[2-methoxyethoxy]phenyl)-3-methylphenyl]-piperidin-1-ylsulphonyl}-2-methylpropanamide; and
N-hydroxy-2-{4-[4-(3-[2-hydroxyethoxy]phenyl)-3-methylphenyl]piperidine-1-ylsulphonyl}-2-methylpropanamide.
In a further aspect, the present invention provides processes for the preparation of a compound of formula (I), or a pharmaceutically or veterinarily acceptable salt thereof, or a pharmaceutically or veterinarily acceptable solvate (including hydrate) of either entity, as illustrated below.
It will be appreciated by persons skilled in the art that, within certain of the processes described, the order of the synthetic steps employed may be varied and will depend inter alia on factors such as the nature of other functional groups present in a particular substrate, the availability of key intermediates and the protecting group strategy (if any) to be adopted. Clearly, such factors will also influence the choice of reagent for use in the said synthetic steps.
Illustrative of protecting group strategies are the synthetic routes to EXAMPLE 64, in which an O-benzyl protected hydroxamate is formed prior to the required Suzuki reaction step, and to EXAMPLE 66, in which alcohol protection using a t-butyldiphenylsilyl group is employed.
It will also be appreciated that various standard substituent or functional group interconversions and transformations within certain compounds of formula (I) will provide other compounds of formula (I). An example is the conversion of the tetrahydropyridine derivative (EXAMPLE 28) to the piperidine derivative (EXAMPLE 29) by hydrogenation.
The following processes are illustrative of the general synthetic procedures which may be adopted in order to obtain the compounds of the invention.
A compound of formula (I) may be prepared directly from an ester of formula (II): 
wherein R5 is C1 to C3 alkyl, and the broken line, A,B, R1, R2, R3, R4, m and n are as previously defined for formula (I), or via the intermediacy of the corresponding carboxylic acid of formula (II) wherein R5 is hydrogen.
When prepared directly from an ester of formula (II), the reaction may be carried out by treatment of the ester with up to a 3-fold excess of hydroxylamine in a suitable solvent at from about room temperature to about 85xc2x0 C. The hydroxylamine is conveniently generated in situ from its hydrochloride salt by conducting the reaction in the presence of a molar equivalent amount of a suitable base such as an alkali metal carbonate or bicarbonate, e.g. potassium carbonate. Preferably the solvent is methanol, optionally combined with tetrahydrofuran or dichloromethane as co-solvent, and the reaction temperature is from about 65 to 70xc2x0 C.
Alternatively, the ester may be converted by conventional hydrolysis to the corresponding carboxylic acid which is then transformed to the required hydroxamic acid of formula (I).
Preferably the hydrolysis is effected under basic conditions using up to about a 6-fold excess of an alkali metal hydroxide in aqueous solution, optionally in the presence of a co-solvent, at from about room temperature to about 85xc2x0 C. Typically the co-solvent is selected from methanol, 1,4-dioxan, a mixture of methanol and tetrahydrofuran and a mixture of methanol and 1,4-dioxan and the reaction temperature is from about 40 to about 70xc2x0 C.
The subsequent coupling step may be achieved using conventional amide-bond forming techniques, e.g. via the acyl chloride derivative and hydroxylamine hydrochloride in the presence of an excess of a tertiary amine such as triethylamine or pyridine to act as acid-scavenger, optionally in the presence of a catalyst such as 4-dimethylaminopyridine, in a suitable solvent such as dichloromethane, at from about 0xc2x0 C. to about room temperature. For convenience, pyridine may also be used as the solvent.
In particular, any one of a host of amino acid coupling variations may be used. For example, the acid of formula (II) wherein R5 is hydrogen may be activated using a carbodiimide such as 1,3-dicyclohexylcarbodiimide or 1-ethyl-3-(3dimethylaminoprop-1-yl)carbodiimide optionally in the presence of 1-hydroxybenzotriazole and/or a catalyst such as 4-dimethylaminopyridine, or by using a halotrisaminophosphonium salt such as bromotris(pyrrolidino)-phosphonium hexafluorophosphate. Either type of coupling is conducted in a suitable solvent such as dichloromethane or dimethylformamide, optionally in the presence of a tertiary amine such as N-methylmorpholine or N-ethyidiisopropylamine (for example when either the hydroxylamine or the activating reagent is presented in the form of an acid addition salt), at from about 0xc2x0 C. to about room temperature. Typically, from 1.1 to 2.0 molecular equivalents of the activating reagent and from 1.0 to 4.0 molecular equivalents of any tertiary amine present are employed.
A preferred reagent for mediating the coupling reaction is O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU).
Preferably a solution of the acid and from 1.0 to 1.2 molecular equivalents of N-ethyidiisopropylamine in a suitable solvent such as anhydrous dimethylformamide or anhydrous 1-methylpyrrolidin-2-one, under nitrogen, is treated with up to a 50% excess of HATU at about room temperature followed, after about 15 to 30 minutes, with up to about a 3-fold excess of hydroxylamine hydrochloride and up to about a 4-fold excess of N-ethyldiisopropylamine, optionally in the same solvent, at the same temperature.
An ester of formula (II) may be prepared from an amine of formula (III): 
wherein the broken line, A,B, R3, R4, m and n are as previously defined for formula (II), by sulphonylation with a compound of formula (IV): 
wherein Z is halo, R5 is C1 to C3 alkyl and R1 and R2 are as previously defined for formula (II). Preferably, Z is chloro.
When R6 is hydrogen, it will normally be advantageous to protect this secondary amino linkage with a conventional amine protecting group.
The reaction may be effected in the presence of up to a 50% excess of an appropriate base in a suitable solvent at from about 0xc2x0 C. to about room temperature. For example, when both R1 and R2 are hydrogen, an appropriate base is 1,8-diazabicyclo[5.4.0]undec-7-ene and a suitable solvent is dichloromethane.
Alternatively, the anion of (III) may be generated initially using up to a 20% excess of a strong base in a suitable solvent, under nitrogen, and then the sulphonylation with from 1.0 to 1.2 molecular equivalents of (IV) effected.
Conveniently, such a coupling may be carried out at room temperature with N,O-bis(trimethylsilyl)acetamide as base and anhydrous tetrahydrofuran as solvent.
Further routes to the preparation of an ester of formula (II), wherein R3 is R7, rely on exploitation of either a Suzuki reaction or a Stille reaction with an ester of formula (II) wherein R3 (but not R4) is either bromo or iodo.
Thus, in the Suzuki reaction, the latter ester is treated with from 1.0 to 1.5 molecular equivalents of a boronic acid of formula R7B(OH)2, in the presence of from 2.0 to 3.0 molecular equivalents of an alkali metal fluoride, about 0.1 molecular equivalents of a triarylphosphine and about 0.05 molecular equivalents of a palladium catalyst in a suitable solvent, under nitrogen, at from about 65 to about 100xc2x0 C. Typically, the fluoride is cesium fluoride, the phosphine is tri-o-tolylphosphine, the catalyst is tris(dibenzylideneacetone)-dipalladium(0) and the solvent is degassed 1,2-dimethoxyethane optionally with 1-methylpyrrolidin-2-one as co-solvent.
In the Stille reaction, the aforementioned ester starting material of formula (II) is treated with from 1.0 to 2.0 molecular equivalents of a suitable trialkylstannane derivative of formula R7Sn(alkyl)3 wherein alkyl is, for example, n-butyl, in the presence of from 2.0 to 3.0 molecular equivalents of a tertiary base, from 0.3 to 0.6 molecular equivalents of a triarylphosphine and from 0.05 to 0.2 molecular equivalents of a palladium catalyst in a suitable solvent, under nitrogen, at from about 65 to about 100xc2x0 C. Typically, the base is triethylamine, the phosphine is tri-o-tolylphosphine, the catalyst is palladium(II) acetate optionally in the presence of tetrakis(triphenylphosphine)palladium(0) and the solvent is anhydrous acetonitrile.
Certain esters of formula (II) wherein at least one of R1 and R2 is other than hydrogen may be conveniently obtained from the xcex1-carbanion of an ester of formula (II) wherein at least one of R1 and R2 is hydrogen by conventional C-alkylation procedures using an alkylating agent of formula (VA) or (VB):
RXxe2x80x83xe2x80x83(VA)
X-W-Yxe2x80x83xe2x80x83(VB)
wherein R is as previously defined for R1 or R2 but is not hydrogen, X and Y may be the same or different and are suitable leaving groups, and W is a C2 to C5 alkylene group which optionally incorporates a heteroatom linkage selected from O, SO, SO2 and NR6 or which is optionally benzo-fused. When R6 is to be hydrogen in a compound of formula (I), then a conventional amine protecting group strategy may be of advantage during this alkylation procedure.
A suitable leaving group may be selected from halo (e.g. chloro, bromo or iodo), C1-C4 alkanesulphonyloxy, trifluoromethanesulphonyloxy and arylsulphonyloxy (e.g. benzenesulphonyloxy or p-toluenesulphonyloxy).
Preferably, X and Y are selected from bromo, iodo and p-toluenesulphonyloxy.
The carbanion may be generated using an appropriate base in a suitable solvent. Typical base-solvent combinations may be selected from lithium, sodium or potassium hydride, lithium, sodium or potassium bis(trimethylsilyl)amide, lithium diisopropylamide and butyllithium, together with toluene, ether, 1,2-dimethoxyethane, tetrahydrofuran, 1,4-dioxan, dimethylformamide, N,N-dimethylacetamide, 1-methylpyrrolidin-2-one and any mixture thereof.
Preferably the base is sodium hydride and the solvent is anhydrous dimethylformamide, optionally with anhydrous tetrahydrofuran as co-solvent, or anhydrous 1-methylpyrrolidin-2-one. For monoalkylation, up to about a 10% excess of base is employed whilst, for dialkylation, from about 2 to about 3 molar equivalents are generally appropriate.
Typically, the carbanion is generated at about room temperature, under nitrogen, and subsequently treated with up to about a 30% excess of the required alkylating agent at the same temperature.
Clearly, when dialkylation is required and R1 and R2 are different, the substituents may be introduced in tandem in a xe2x80x9cone-pot reactionxe2x80x9d or in separate steps.
A particularly convenient, alternative alkylation method involves treatment of the substrate with the required alkylating agent in the presence of from 3.0 to 3.5 molecular equivalents of anhydrous potassium carbonate in anhydrous dimethyl sulphoxide or anhydrous 1,2-dimethoxyethane, under nitrogen, at about room temperature.
Clearly, an alternative variation for preparing a compound of formula (II) is to introduce R1 and/or R2 into a suitable bromo or iodo intermediate before further elaboration via, for example, a Suzuki or Stille reaction.
An amine of formula (III) may be obtained by standard chemical procedures. For example, when B is absent, m is 2 and n is 1, a suitably N-protected piperidin-4-one of formula (VI): 
wherein P is a conventional amine protecting group, is reacted with a carbanion derivative of a compound of formula (VII): 
wherein Z is as previously defined for formula (IV) and R3 and R4 are as previously defined for formula (III), to provide a compound of formula (VIII): 
Preferably, Z is chloro, bromo or iodo.
Conveniently (VII) is converted to an aryllithium or aryl Grignard derivative whilst, of the plethora of amine protecting groups available, P is typically t-butoxycarbonyl (Boc) or benzyl.
When P is Boc, (VIII) may be transformed directly to a compound of formula (III), wherein the broken line represents a bond, A is C, B is absent, m is 2, n is 1 and R3 and R4 are as previously defined for formula (III), using trifluoroacetic acid optionally in a suitable solvent such as dichloromethane at about room temperature. Alternatively, when P is benzyl, (VIII) may be converted in two steps to the same compound of formula (III). For example, in the first step, dehydration may be effected in refluxing toluene using p-toluenesulphonic acid and a Dean-Stark apparatus. N-Deprotection of the resulting alkene (1,2,3,6-tetrahydropyridine derivative), in the second step, may be achieved using 1-chloroethyl chloroformate in refluxing toluene followed by treatment of the reaction mixture, at room temperature, with either methanol or ethanol.
This unsaturated piperidine may be converted to a compound of formula (III) wherein the broken line does not represent a bond, A is CH, B is absent, m is 2, n is 1 and R3 and R4 are as previously defined for formula (III) under conventional catalytic, or catalytic transfer, hydrogenation conditions. Alternatively, these hydrogenation conditions may be employed to convert the previously described N-benzyl alkene (1,2,3,6-tetrahydropyridine derivative) to the same piperidine derivative, directly in one step. Furthermore, this fully saturated piperidine is also available in one step from (VIII) when P is Boc by standard ionic hydrogenation using, for example, triethylsilane and trifluoroacetic acid in dichloromethane.
Other amines of formula (III), when neither commercially available nor subsequently described, can be obtained either by analogy with the processes described in the Preparations section or by conventional synthetic procedures, in accordance with standard textbooks on organic chemistry or literature precedent, from readily accessible starting materials using appropriate reagents and reaction conditions.
Moreover, persons skilled in the art will be aware of variations of, and alternatives to, those processes described hereinafter in the EXAMPLEs and Preparations sections which allow the compounds defined by formula (I) to be obtained.
The pharmaceutically and veterinarily acceptable base salts of the compounds of formula (I) may also be prepared in a conventional manner. For example a solution of the hydroxamic acid is treated with the appropriate base, either neat or in a suitable solvent, and the resulting salt isolated either by filtration or by evaporation under vacuum of the reaction solvent. Pharmaceutically and veterinarily acceptable acid addition salts can be obtained in an analogous manner by treating a solution of a basic compound of formula (I) with the appropriate acid. Both types of salt may be formed or interconverted using ion-exchange resin techniques.
The biological activities of the compounds of the present invention were determined by the following test methods, which are based on the ability of the compounds to inhibit the cleavage of various fluorogenic peptides by MMPs 1, 2, 3, 9, 13 and 14.
The assays for MMPs 2, 3, 9 and 14 are based upon the original protocol described in FEBS, 1992, 296, 263, with the minor modifications described below.
Inhibition of MMP-1
Enzyme Preparation
Catalytic domain MMP-1 was prepared in Pfizer Central Research laboratories. A stock solution of MMP-1 (1 xcexcM) was activiated by the addition of aminophenylmercuric acetate (APMA), at a final concentration of 1 mM, for 20 minutes at 37xc2x0 C. MMP-1 was then diluted in Tris-HCl assay buffer (50 mM Tris, 200 mM NaCl, 5 mM CaCl2, 20 xcexcM ZnSO4 and 0.05% Brij 35, pH 7.5) to a concentration of 10 nM. The final concentration of enzyme used in the assay was 1 nM.
Substrate
The fluorogenic substrate used in this assay was Dnp-Pro-xcex2-cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-Lys-(N-Me-Ala)-NH2 as originally described in Anal. Biochem., 1993, 212, 58. The final substrate concentration used in the assay was 10 xcexcM.
Determination of Enzyme Inhibition
The test compound was dissolved in dimethyl sulphoxide and diluted with assay buffer so that no more than 1% dimethyl sulphoxide was present. Test compound and enzyme were added to each well of a 96 well plate and allowed to equilibrate for 15 minutes at 37xc2x0 C. in an orbital shaker prior to the addition of substrate. Plates were then incubated for 1 hour at 37xc2x0 C. prior to determination of fluorescence (substrate cleavage) using a fluorimeter (Fluostar; BMG Lab Technologies, Aylesbury, UK) at an excitation wavelength of 355 nm and emission wavelength of 440 nm. The potency of inhibition was measured from the amount of substrate cleavage obtained using a range of test compound concentrations and, from the resulting dose-response curve, an IC50 value (the concentration of inhibitor required to inhibit 50% of the enzyme activity) was calculated.
Inhibition of MMP-2, MMP-3 and MMP-9
Enzyme Preparation
Catalytic domains MMP-2, MMP-3 and MMP-9 were prepared in Pfizer Central Research laboratories. A stock solution of MMP-2, MMP-3 or MMP-9 (1 xcexcM) was activated by the addition of APMA. For MMP-2 and MMP-9, a final concentration of 1 mM APMA was added, followed by incubation for 1 hour at 37xc2x0 C. MMP-3 was activated by the addition of 2 mM APMA, followed by incubation for 3 hours at 37xc2x0 C. The enzymes were then diluted in Tris-HCl assay buffer (100 mM Tris, 100 mM NaCl, 10 mM CaCl2 and 0.16% Brij 35, pH 7.5) to a concentration of 10 nM. The final concentration of enzyme used in the assays was 1 nM.
Substrate
The fluorogenic substrate used in this screen was Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NH2 (Bachem Ltd., Essex, UK) as originally described in J.Biol.Chem., 1994, 269, 20952. This substrate was selected because it has a balanced hydrolysis rate against MMPs 2, 3 and 9 (kcat/km of 54,000, 59,400 and 55,300 sxe2x88x921 Mxe2x88x921 respectively). The final substrate concentration used in the assay was 5 xcexcM.
Determination of Enzyme Inhibition
The test compound was dissolved in dimethyl sulphoxide and diluted with assay buffer so that no more than 1% dimethyl sulphoxide was present. Test compound and enzyme were added to each well of a 96 well plate and allowed to equilibrate for 15 minutes at 37xc2x0 C. in an orbital shaker prior to the addition of substrate. Plates were then incubated for 1 hour at 37xc2x0 C., prior to determination of fluorescence using a fluorimeter (Fluostar; BMG Lab Technologies, Aylesbury, UK) at an excitation wavelength of 328 nm and emission wavelength of 393 nm. The potency of inhibition was measured from the amount of substrate cleavage obtained using a range of test compound concentrations and, from the resulting dose-response curve, an IC50 value (the concentration of inhibitor required to inhibit 50% of the enzyme activity) was calculated.
Inhibition of MMP-13
Enzyme Preparation
Human recombinant MMP-13 was prepared by PanVera Corporation (Madison, Wis.) and characterised at Pfizer Central Research laboratories. A 1.9 mg/ml stock solution was activated with 2 mM APMA for 2 hours at 37xc2x0 C. MMP-13 was then diluted in assay buffer (50 mM Tris, 200 mM NaCl, 5 mM CaCl2, 20 xcexcM ZnCl2 and 0.02% Brij 35, pH 7.5) to a concentration of 5.3 nM. The final concentration of enzyme used in the assay was 1.3 nM.
Substrate
The fluorogenic substrate used in this screen was Dnp-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(NMA)-NH2. The final substrate concentration used in the assay was10 xcexcM.
Determination of Enzyme Inhibition
The test compound was dissolved in dimethyl sulphoxide and diluted with assay buffer so that no more than 1% dimethyl sulphoxide was present. Test compound and enzyme were added to each well of a 96 well plate. The addition of substrate to each well initiated the reaction. Fluorescence intensity was determined using a 96 well plate fluorimeter (Cytofluor II; PerSeptive Biosystems, Inc., Framingham, Mass.) at an excitation wavelength of 360 nm and emission wavelength of 460 nm. The potency of inhibition was measured from the amount of substrate cleavage obtained using a range of test compound concentrations and, from the resulting dose-response curve, an IC50 value (the concentration of inhibitor required to inhibit 50% of the enzyme activity) was calculated.
Inhibition of MMP-14
Enzyme Preparation
Catalytic domain MMP-14 was prepared in Pfizer Central Research laboratories. A 10 xcexcM enzyme stock solution was activated for 20 minutes at 25xc2x0 C. following the addition of 5 xcexcg/ml of trypsin (Sigma, Dorset, UK). The trypsin activity was then neutralised by the addition of 50 xcexcg/ml of soyabean trypsin inhibitor (Sigma, Dorset, UK), prior to dilution of this enzyme stock solution in Tris-HCl assay buffer (100 mM Tris, 100 mM NaCl, 10 mM CaCl2, 0.16% Brij 35, pH 7.5) to a concentration of 10 nM. The final concentration of enzyme used in the assay was 1 nM.
Substrate
The fluorogenic substrate used in this screen was Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (Bachem Ltd., Essex, UK) as described in J.Biol.Chem., 1996, 271, 17119.
Determination of Enzyme Inhibition
This was performed as described for MMPs 2, 3 and 9.
In human therapy, the compounds of formula (I), their pharmaceutically acceptable salts, and pharmaceutically acceptable solvates of either entity, can be administered alone, but will generally be administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
They may be administered orally in the form of tablets containing such excipients as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solution or suspensions containing flavouring or colouring agents. They can also be injected, for example intravenously, intramuscularly or subcutaneously, and are best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccarides to make the solution isotonic with blood. For other routes of parenteral administration, such as buccal or sublingual, they may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
In addition, the compounds and their salts may be administered topically in the form of sterile creams, gels, suspensions, lotions, ointments, dusting powders, sprays, drug-incorporated dressings or via a skin patch. For example, they can be incorporated into a cream consisting of an aqueous or oily emulsion of polyethylene glycols or liquid paraffin, or they can be incorporated into an ointment consisting of a white wax soft paraffin base, or as a hydrogel with cellulose or polyacrylate derivatives or other viscosity modifiers, or as a dry powder or liquid spray or aerosol with butane/propane, HFA or CFC propellants, or as a drug-incorporated dressing either as a tulle dressing, with white soft paraffin or polyethylene glycol impregnated gauze dressings or with hydrogel, hydrocolloid, alginate or film dressings. Moreover, the compounds and salts may be administered intraocularly as an eye drop with appropriate buffers, viscosity modifiers (e.g. cellulose derivatives), preservatives (e.g. benzalkonium chlorides (BZK)) and agents to adjust tonicity (e.g. sodium chloride).
All such formulations may also contain stabilisers and preservatives.
Depending on the route of administration to human patients, the daily dosage level of the compounds of formula (I) and their salts may be from 0.001 to 20 mg/kg, in single or divided doses. Thus, for example, tablets or capsules could contain from 0.02 to 500 mg of active compound for administration singly or two or more at a time as appropriate.
The physician in any event will determine the actual dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case; there can of course be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.
For veterinary use, a compound of formula (I), or a veterinarily acceptable salt thereof, or a veterinarily acceptable solvate of either entity, is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.
Thus the invention provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of either entity, together with a pharmaceutically acceptable diluent or carrier.
It further provides a veterinary formulation comprising a compound of formula (I), or a veterinarily acceptable salt thereof, or a veterinarily acceptable solvate of either entity, together with a veterinarily acceptable diluent or carrier.
The invention also provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of either entity, or a pharmaceutical composition containing any of the foregoing, for use as a human medicament.
In addition, it provides a compound of formula (I), or a veterinarily acceptable salt thereof, or a veterinarily acceptable solvate of either entity, or a veterinary formulation containing any of the foregoing, for use as an animal medicament.
In yet another aspect, the invention provides the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of either entity, for the manufacture of a human medicament for the curative or prophylactic treatment of a medical condition for which a MMP inhibitor is indicated.
It also provides the use of a compound of formula (I), or a veterinarily acceptable salt thereof, or a veterinarily acceptable solvate of either entity, for the manufacture of an animal medicament for the curative or prophylactic treatment of a medical condition for which a MMP inhibitor is indicated.
Moreover, the invention provides the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate containing either entity, for the manufacture of a human medicament for the curative or prophylactic treatment of atherosclerotic plaque rupture, myocardial infarction, heart failure, restenosis, stroke, periodontal disease, tissue ulceration, wound repair, skin diseases, cancer metastasis, tumour angiogenesis, age-related macular degeneration, fibrotic disease, rheumatoid arthritis, osteoarthritis and inflammatory diseases dependent on migratory inflammatory cells.
It also provides the use of a compound of formula (I), or a veterinarily acceptable salt thereof, or a veterinarily acceptable solvate containing either entity, for the manufacture of an animal medicament for the curative or prophylactic treatment of atherosclerotic plaque rupture, myocardial infarction, heart failure, restenosis, stroke, periodontal disease, tissue ulceration, wound repair, skin diseases, cancer metastasis, tumour angiogenesis, age-related macular degeneration, fibrotic disease, rheumatoid arthritis, osteoarthritis and inflammatory diseases dependent on migratory inflammatory cells.
Additionally, the invention provides a method of treating or preventing a medical condition for which a MMP inhibitor is indicated, in a mammal (including a human being), which comprises administering to said mammal a therapeutically effective amount of a compound of formula (I), or a pharmaceutically or veterinarily acceptable salt thereof, or a pharmaceutically or veterinarily acceptable solvate of either entity, or a pharmaceutical composition or veterinary formulation containing any of the foregoing.
Still further, the invention provides a method of treating or preventing atherosclerotic plaque rupture, myocardial infarction, heart failure, restenosis, stroke, periodontal disease, tissue ulceration, wound repair, skin diseases, cancer metastasis, tumour angiogenesis, age-related macular degeneration, fibrotic disease, rheumatoid arthritis, osteoarthritis and inflammatory diseases dependent on migratory inflammatory cells, in a mammal (including a human being), which comprises administering to said mammal a therapeutically effective amount of a compound of formula (I), or a pharmaceutically or veterinarily acceptable salt thereof, or a pharmaceutically or veterinarily acceptable solvate of either entity, or a pharmaceutical composition or veterinary formulation containing any of the foregoing.
The invention also includes any novel intermediates described herein, for example those of formula (II).
The syntheses of the compound of the invention and of the intermediates for use therein are illustrated by the following EXAMPLEs and Preparations.
Room temperature means 20 to 25xc2x0 C.
Flash chromatography refers to column chromatography on silica gel (Kieselgel 60, 230-400 mesh).
Melting points are uncorrected.
1H Nuclear magnetic resonance (NMR) spectra were recorded using a Bruker AC300, a Varian Unity Inova-300 or a Varian Unity Inova-400 spectrometer and were in all cases consistent with the proposed structures. Characteristic chemical shifts (xcex4) are given in parts-per-million downfield from tetramethylsilane using conventional abbreviations for designation of major peaks: e.g. s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad.
Mass spectra were recorded using a Finnigan Mat. TSQ 7000 or a Fisons Intruments Trio 1000 mass spectrometer. LRMS means low resolution mass spectrum and the calculated and observed ions quoted refer to the isotopic composition of lowest mass.